1ST CUNEO CITY IMMUNOTHERAPY CONFERENCE 17-18 Maggio 2018 PASSIVE IMMUNOTHERAPY ANTIBODY-DRUG CONJUGATES GEMTUZUMAB OZOGAMICIN Do#. Filippo Gherlinzoni Direore Unità Operava Complessa di Ematologia Ospedale “Ca’ Foncello” - Treviso IL RIECCOLO Blood Reviews 28 (2014) 143–153 Contents lists available at ScienceDirect Blood Reviews journal homepage: www.elsevier.com/locate/blre REVIEW The past and future of CD33 as therapeutic target in acute myeloid leukemia a a,b a,b,c, George144 S. Laszlo , Elihu H. Estey , Roland B. Walter ⁎G.S. Laszlo et al. / Blood Reviews 28 (2014) 143–153 a Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA b Department of Medicine, Division of Hematology, University of Washington, Seattle, WA, USA c Department of Epidemiology, University of Washington, Seattle, WA, USA as cytokine-induced cellular proliferation [14,33]. Engagement with CD33 antibodies has similarly been shown to affect cytokine and article info abstract chemokine secretion by monocytes, although both increases [34] or Keywords: CD33 is a myeloid differentiation antigen with endocytic properties.decreases It is broadly[33] expressedhave on been acute myeloidobserved in vitro. Acute myeloid leukemia leukemia (AML) blasts and, possibly, some leukemic stem cells and has therefore been exploited as target for Antibody therapeutic antibodies for many years. The improved survival seen in many patients when the antibody-drug Antibody-drug conjugate conjugate, gemtuzumab ozogamicin, is added to conventional chemotherapy validates this approach. However, fi 2.3. Endocytic properties of CD33 Bispeci c antibody many attempts with unconjugated or conjugated antibodies have been unsuccessful, highlighting the challenges BiTE of targeting CD33 in AML. With the development of improved immunoconjugates and CD33-directed strategies CD33 fi Chimeric antigen receptor that harness immune effector cells, therapeutics with enhanced ef cacy may soon become available. Toxic effects A therapeuticallyfi important characteristic of CD33 is its internaliza- Gemtuzumab ozogamicin on normal hematopoietic cells may increase in parallel with this increased ef cacy and demand new supportive Immunotherapy care measures, including possibly rescue with donor cells, to minimizetion when morbidity bound and mortality by bivalent from drug- antibodies; this process is slower than that Radioimmunoconjugate induced cytopenias and to optimize treatment outcomes with these agents in patients with AML. observed© 2014 Elsevier with other Ltd. All rights cell surface reserved. antigens such as the transferrin recep- tor [35–43]. Mechanistic studies indicate that endocytosis is largely lim- ited and determined by the intracellular domain of CD33 and may be regulated by tyrosine phosphorylation and perhaps ubiquitylation of 1. Introduction 2. Physiologic characteristics of CD33 the cytoplasmic tail, although some internalization of CD33/antibody Since the invention of hybridoma technology 4 decades ago, mono- CD33 is a member of the sialiccomplexes acid-binding occursimmuno in aglobulin-like phosphorylation-independent manner [41–43]. clonal antibodies have revolutionized the care of patients with cancer. lectins (Siglecs), a discrete subset of the immunoglobulin (Ig) super- An increasing number of unconjugated, toxin-loaded, and radiolabeled family molecules (Fig. 1) [4,5]Related. This 67kD to single this pass endocytic transmembrane property, engagement of CD33 with bivalent antibodies have shown anti-tumor efficacy and have been approved glycoprotein is characterized byantibodies an amino-terminal leads V-set to a Ig-like decrease domain (“modulation”) of CD33 cell surface levels for indications in an expanding list of malignancies, including acute that mediates sialic acid binding and a C2-set Ig-like domain in its extra- myeloid leukemia (AML) [1,2]. AML has been a paradigm for the thera- cellular portion [6–8]. Alternative[35,40,44] splicing. Although of CD33 RNA new leads CD33 to a sites are continuously expressed [40], peutic use of monoclonal antibodies, in no small part because malignant shorter isoform that is expressedthis on feature the cell surface. could This reduce isoform the lacks efficacy of CD33-directed therapeutics cells are readily accessible and express well-defined cell surface anti- the V-set Ig-like domain as well as the disulfide bond linking the gens. Most efforts to date have focused on exploiting CD33 as a target V- and C2-set Ig-like domains.because While the of biological the reduction relevance of of available this target binding sites. in this disease, and the CD33-directed immunoconjugate, gemtuzumab splicing process is unknown, it may be important for the development ozogamicin (GO), was the first anti-cancer antibody-drug conjugate to and use of CD33-directed drugs. Specifically, a dominant epitope, recog- obtain marketing approval in the U.S. [3] Still, targeting CD33 has nized by the majority of initial3. CD33 CD33 antibodies, in AML is located and other on the malignanciesV-set proven challenging, as perhaps best reflected by the eventual market Ig-like domain. Thus, some CD33 antibody-based therapeutics will only withdrawal of GO because of concerns over excess toxicity and lack of recognize the full-length but not the shorter splice isoform of CD33 fi efficacy. In this article, we will summarize the biologic characteristics [9,10]. Depending on how antigen positivity is de ned, CD33 is found on at of CD33, emphasizing the properties that make it appealing as a thera- The cytoplasmic tail of CD33least contains a subset 2 conserved of blasts tyrosine-based in nearly all AML patients [45,46], consistent with peutic target, appraise attempts made thus far with CD33-directed ther- inhibitory signaling motifs, which, upon phosphorylation by Src family its characteristic as a myeloid differentiation antigen. Although surface apies, andFig. discuss 1. Structure current and of CD33. future Scheme therapeutic depicting directions the domain in this fi structureeld. kinases, of CD33 provide as well docking as indi- sites for the recruitment and activation of Src Laszlo G.S. et al Blood Reviews 2014, 143-153 homology-2 (SH2) domain-containing tyrosine phosphatases such as vidual amino acids that have been implicated in phosphorylation or ubiquitylation events. levels show considerable inter-patient variability (N2-log fold) [15,45, SHP-1 and SHP-2. While the signaling events downstream of CD33 4 Abbreviations: C2, C2-set Ig-like domain; P, phospho-; SFKs, Src-familyremain kinases; poorly understood,Ub, ubiq- these46] phosphatases, CD33 expression may not only is dephos- relatively limited with an average of ~10 ⁎ Corresponding author at: Clinical Research Division, Fred Hutchinson Cancer Research uitin; V, V-set Ig-like domain. phorylate CD33 as part of a negative feedback loop but also dephos- Center; 1100 Fairview Ave N, D2-190; Seattle, WA 98109-1024, USA. Tel.: +1 206 667 molecules/AML blast [15,47] and is typically even lower in immature 3599; fax: +1 206 667 5255. phorylate and negatively regulate nearby receptors+ [11- –13]. Through+ + - E-mail address: [email protected] (R.B. Walter). its SH2 domain, suppressor(e.g. of cytokine CD34 signaling/CD38 3/CD123 (SOCS3) canor CD34 /CD38 ) cell subsets [46,48]. compete with SHP-1 or SHP-2 for binding to phosphorylated CD33 and CD33 expression correlates with disease characteristics: for example, http://dx.doi.org/10.1016/j.blre.2014.04.001recruit the ECS (Elongin B/C-Cul2/Cul5-SOCS-box protein) E3 ubiquitin expression is homogeneous and typically bright in acute promyelocytic 0268-960X/©ligase 2014 Elsevier complex, Ltd. All rights which reserved. ultimately leads to ubiquitylation and results in leukemias (APL) [49,50]. High levels of CD33 are also associated with accelerated proteasomal degradation of CD33 (Fig. 1) [14]. NPM1 as well as high allelic FLT3/ITD mutations, while expression is generally low with core-binding factor translocations [45,46,51]. Likely 2.1. CD33 as myeloid differentiation antigen at least partially related to such associations, it has been a recurrent observation in pediatric trials that, on average, patients whose AML In healthy individuals, CD33 is primarily a myeloid differentiation blasts display high CD33 levels experience inferior disease-free and antigen with initial expression at the very early stages of myeloid cell overall survival when treated with conventional chemotherapy that development: it is found on normal multipotent myeloid precursors, does not include CD33-targeted agents [52,53]; similar data from unipotent colony-forming cells, and maturing granulocytes and mono- adult patients is currently not available. In AML patients, CD33 can cytes. On CD34+/CD33+ bone marrow cells, CD33 expression has also be detected as soluble protein in the circulation and may provide been estimated to average around 8 × 103 molecules/cell, although some prognostic information [54,55]; however, its role as predictive levels vary widely (1–20 × 103 molecules/cell) [15]. Expression is biomarker has not been studied in detail. It is also unclear as to what down-regulated to lower levels on neutrophils (~2–2.5 × 103 mole- degree, if any, soluble CD33 might interfere with the therapeutic effica- cules/cell) and macrophages but retained on circulating monocytes cy of CD33 antibodies, although some in vitro evidence suggests that and dendritic cells [15–20]. CD33 can also be displayed on subsets of soluble CD33 may not impact the activity of CD33-targeted immuno-